Identification of 3-ketosteroid 9-alfa-hydroxylase genes and microorganisms blocked in 3-ketosteroid 9-alfa-hydroxylase activity

ABSTRACT

The invention relates to an isolated polynucleotide sequence comprising a nucleic acid sequence encoding the amino acid sequence of KshA protein or of KshB protein, encoded by nucleotides 499-1695 of SEQ ID NO:1 or by nucleotides 387-1427 of SEQ ID NO:2, respectively, and functional homologues thereof. The polynucleotides of the invention can be used to construct genetically modified microorganisms blocked in 3-ketosteroid 9α-hydroxylase activity, which are useful in the microbial degradation of steroids to accumulate certain steroid products.

The invention relates to isolated polynucleotide sequences encoding3-ketosteroid 9α-hydroxylase components, to microorganisms blocked in3-ketosteroid 9α-hydroxylase activity, to a method for the preparationof said microorganisms, and to the use of such microorganisms in steroidΔ¹-dehydrogenation.

To date very limited knowledge is available on 3-ketosteroid9α-hydroxylase (KSH), the enzyme performing the 9α-hydroxylation of4-androstene-3,17-dione (AD) and 1,4-androstadiene-3,17-dione (ADD) inmicrobial sterol/steroid degradation. No nucleotide sequences of thegenes encoding KSH components have been reported. Furthermore,difficulties are faced during enzyme purification procedures (Chang, F.N. et al. Biochemistry (1964) 3:1551-1557; Strijewski, A. Eur. J.Biochem. (1982) 128:125-135). A three-component monooxygenase with KSHactivity has been partially purified from Aocardia sp. M117 and wasfound to constitute a three-component enzyme system, composed of aflavoprotein reductase and two ferredoxin proteins (Strijewski, A. Eur.J. Biochem. (1982) 128:125-135). In Arthrobacter oxydans 317,9α-hydroxylation of the steroid poly-cyclic ring structure appearedplasmid-borne (Dutta, R. K. et al. J. Basic Microbiol. (1992)32:317-324). Nucleotide sequence analysis of the plasmid, however, wasnot reported.

The lack of genetic data has hampered the construction of molecularlydefined mutant strains with desired properties (i.e. blocked9α-hydroxylation of steroids) by genetic engineering. Mutants have beenisolated by classical mutagenesis, but these strains usually areinadequate in industrial processes mostly due to genetic instabilityand/or low bioconversion efficiencies. Molecularly defined mutants haveadvantages compared to mutants generated by classical mutagenesis. Theconstructed mutants are genetically stable and the introduced mutationsare well-defined genetic modifications. Construction of geneticallyengineered strains make the widespread use of chemical agents to block9α-hydroxylation (e.g. α,α-dipyridyl, o-phenanthroline) obsolete.Chemical agents used to block KSH activity mostly are not reactionspecific and inhibit other important enzymatic reactions (e.g. sterol26-hydroxylation in sterol side chain degradation), which may havenegative effects on sterol bioconversion efficiency. The use of definedmutants by genetic engineering overcomes these problems.

3-Ketosteroid 9α-hydroxylase (KSH) is a key-enzyme in the microbialsteroid ring B-opening pathway. KSH catalyzes the conversion of AD into9α-hydroxy-4-androstene-3,17-dione (9OHAD) and ADD into the chemicallyunstable compound [9OHADD]. KSH activity has been found in manybacterial genera (Martin, C. K. A. Adv. Appl. Microbiol. (1977) 22:29-58; Kieslich, K. J Basic Microbiol. (1985) 25: 461-474; Mahato, S. B.et al. Steroids (1997) 62: 332-345): e.g. Rhodococcus (Datcheva, V. K.et al. Steroids (1989) 54:271-286; Van der Geize et al. FEMS Microbiol.Lett. (2001) 205:197-202, Nocardia(Strijewski, A. Eur. J. Biochem.(1982) 128:125-135), Arthrobacter (Dutta, R. K. et al. J. BasicMicrobiol. (1992) 32:317-324) and Mycobacterium (Wovcha, M. G. et al.Biochim Biophys Acta (1978) 531:308-321). Bacterial strains lacking KSHactivity are being considered important in sterol/steroidbiotransformation. Mutants blocked in KSH activity will be able toperform only the KSTD (3-ketosteroid Δ¹-dehydrogenase) reaction, therebyallowing selective Δ¹-dehydrogenation of steroid compounds. Examples arethe cortisol biotransformation into prednisolone and the ADbiotransformation into ADD. Sterol bioconversion by mutants blocked atthe level of steroid 9α-hydroxylation may also carry out a selectivedegradation of the sterol side chain thereby accumulating AD and/or ADDwhich are excellent precursors for the synthesis of bioactive steroidhormones.

According to one aspect of the present invention, the isolatedpolynucleotide sequences of two genes, designated kshA and kshB ofRhodococcus erythropolis are now provided: SEQ ID NO:1 and SEQ ID NO:2,respectively. KshA protein is encoded by nucleotides 499-1695 of SEQ IDNO:1 and KshB protein by nucleotides 387-1427 of SEQ ID NO:2. Thus, inparticular preferred are polynucleotides comprising the complete codingDNA sequences of the nucleotides 499-1695 of SEQ ID NO:1 and of thenucleotides 387-1427 of SEQ ID NO:2, respectively.

Furthermore, to accommodate codon variability the invention alsoincludes sequences coding for the same amino acid sequences of the KshAprotein and the KshB protein. Also portions of the coding sequencescoding for individual domains of the expressed protein are part of theinvention as well as allelic and species variations thereof. Sometimes,a gene is expressed as a splicing variant, resulting in the inclusion ofan additional exon sequence, or the exclusion of an exon. Also a partialexon sequence may be included or excluded. A gene may also betranscribed from alternative promotors that are located at differentpositions within a gene, resulting in transcripts with different 5′ends. Transcription may also terminate at different sites, resulting indifferent 3′ ends of the transcript. These sequences as well as theproteins encoded by these sequences all are expected to perform the sameor similar functions and form also part of the invention. The sequenceinformation as provided herein should not be so narrowly construed as torequire inclusion of erroneously identified bases. The specific sequencedisclosed herein can be readily used to isolate the complete genes whichin turn can easily be subjected to further sequence analyses therebyidentifying sequencing errors.

The present invention further relates to polynucleotides having slightvariations or having polymorphic sites. Polynucleotides having slightvariations encode polypeptides which retain the same biological functionor activity as the natural, mature protein.

The DNA according to the invention may be obtained from cDNA usingsuitable probes derived from SEQ ID NO:1 or SEQ ID NO:2. Alternatively,the coding sequence might be genomic DNA, or prepared using DNAsynthesis techniques. The polynucleotide may also be in the form of RNA.If the polynucleotide is DNA, it may be in single stranded or doublestranded form. The single strand might be the coding strand or thenon-coding (anti-sense) strand.

The present invention further relates to polynucleotides which have atleast 70%, preferably 80%, more preferably 90%, even more preferred 95%,and highly preferably 98% and most preferred at least 99% identity withthe entire DNA sequence of the nucleotides 499-1695 of SEQ ID NO:1 andof the nucleotides 387-1427 of SEQ ID NO:2, respectively. Suchpolynucleotides encode polypeptides which retain the same biologicalfunction or activity as the natural, mature protein. Alternatively, alsofragments of the above mentioned polynucleotides which code for domainsof the protein which still are capable of binding to substrates areembodied in the invention.

The percentage of identity between two sequences can be determined withprograms such as Clustal W 1.7 (Thompson J. D., et al. Nucleic AcidsRes. (1994) 22:4673-4680: “CLUSTALW: improving the sensitivity ofprogressive multiple sequence alignment through sequence weighing,position-specific gap penalties and weight matrix.”) used in defaultsettings. The percentage identity generally is defined by the number ofidentical residues between the two sequences divided by the total numberof residues of the known sequence.

Similarity is defined as a combination of identity together with allsemi-conserved amino acid residues in the alignment according to thegroups as defined in ClustalW 1.7:

‘*’=identity=indicates positions which have a single, fully conservedresidue

‘:’=semi-conserved=indicates that one of the following ‘strong’ groupsis fully conserved. STA, NEQK, NHQK, NDEQ, QHRK, MILV, MILF, HY, FYW.

‘.’=semi-conserved=indicates that one of the following ‘weaker’ groupsis fully conserved. CSA, ATV, SAG, STNK, STPA, SOND, SNDEQK, NDEQHK,NEQHRK, FVLIM, HFY.

These are all the positively scoring groups that occur in the GonnetPam250 matrix.

Also within the scope of this invention are functional homologues of thenew genes e.g. in the family of Actinomycetales (e.g. Rhodococcus,Nocardia, Arthrobacter, Corynebacterium and Mycobacterium).

In order to identify such genes with similar action in othermicroorganisms, any method for detection of (poly)nucleotides known inthe art for such purpose is included herewith. For example, nucleotideelongation methods/amplification methods may be considered, but also,such method may comprise the steps of: hybridizing to a sample a probespecific for a polynucleotide encoding an amino acid sequence of KshA orKshB under conditions effective for said probe to hybridize specificallyto said polynucleotide and determining the hybridization of said probeto polynucleotides in said sample. The term “specific” in this respectmeans that the majority of hybridization takes place with apolynucleotide of this invention. Preferably, said probe comprises atleast 25 of the nucleotides of SEQ ID NO:1 or SEQ ID NO:2. Morepreferred, the probe comprises 50, and in particular preferred 10 morethan 100, nucleotides of SEQ ID NO:1 or SEQ ID NO:2. Most preferred, theprobe consists of a polynucleotide of nucleotides selected from thenucleotides 499-1695 of SEQ ID NO:1 and of the nucleotides 387-1427 ofSEQ ID NO:2, respectively. Appropriate stringency conditions whichpromote DNA hybridization, for example, 6.0× sodium chloride/sodiumcitrate (SSC) at about 45° C., followed by a wash of 2.0×SSC at 50° C.,are known to those skilled in the art or can be found in CurrentProtocols in Molecular Biology, John Wiley & Sons, N.Y. (1989),6.3.1-6.3.6. For example, the salt concentration in the wash step can beselected from low stringency of about 2.0×SSC at 50° C. to a highstringency of about 0.2×SSC at 50° C. In addition, the temperature inthe wash step can be increased from low stringency conditions at roomtemperature, about 22° C., to high stringency conditions at about 65° C.

Alternatively, the polynucleotides of this invention may also be usedfor targeting specific genes, e.g. for the purpose of gene disruption inother species (see for example WO 01/31050 and references citedtherein).

The sequence of the newly identified polynucleotide of the presentinvention, SEQ ID NO:1 and SEQ ID NO:2 may also be used in thepreparation of vector molecules for the expression of the encodedprotein in suitable host cells. A wide variety of host cell and cloningvehicle combinations may be usefully employed in cloning the nucleicacid sequences coding for the proteins KshA or KshB or parts thereof.For example, useful cloning vehicles may include chromosomal,non-chromosomal and synthetic DNA sequences such as various knownbacterial plasmids and wider host range plasmids and vectors derivedfrom combinations of plasmids and phage or virus DNA. Vehicles for usein expression of the polynucleotides of the present invention or a partthereof comprising a functional domain will further comprise controlsequences operably linked to the nucleic acid sequence coding for theprotein. Such control sequences generally comprise a promoter sequenceand sequences which regulate and/or enhance expression levels. Of coursecontrol and other sequences can vary depending on the host cellselected.

Suitable expression vectors are for example bacterial or yeast plasmids,wide host range plasmids and vectors derived from combinations ofplasmid and phage or virus DNA. Vectors derived from chromosomal DNA arealso included. Furthermore an origin of replication and/or a dominantselection marker can be present in the vector according to theinvention. The vectors according to the invention are suitable fortransforming a host cell. Integrative vectors may also be regarded assuitable expression vehicles.

Recombinant expression vectors comprising DNA of the invention as wellas cells transformed with said DNA or said expression vector also formpart of the present invention.

Suitable host cells according to the invention are bacterial host cells,yeast and other fungi, insect, plant or animal host cells such asChinese Hamster Ovary cells or monkey cells or human cell lines. Thus, ahost cell which comprises DNA or expression vector according to theinvention is also within the scope of the invention. The engineered hostcells can be cultured in conventional nutrient media which can bemodified e.g. for appropriate selection, amplification or induction oftranscription. The culture conditions such as temperature, pH, nutrientsetc. are well known to those ordinary skilled in the art.

The techniques for the preparation of DNA or the vector according to theinvention as well as the transformation or transfection of a host cellwith said DNA or vector are standard and well known in the art, see forinstance Sambrook et al., Molecular Cloning: A laboratory Manual. 2ndEd., Cold Spring Harbor Laboratory, Cold Spring Harbor, N.Y., 1989.

In another aspect of the invention, there is provided for a proteincomprising the amino acid sequence encoded by any of the above describedDNA molecules. Preferably, the protein according to the inventioncomprises an amino acid sequence encoded by the nucleotides 499-1695 ofSEQ ID NO:1 or of the nucleotides 387-1427 of SEQ ID NO:2, respectively.Also part of the invention are proteins resulting from posttranslational processing, which proteins are encoded by thepolynucleotide of this invention.

Also functional equivalents, that is proteins homologous to amino acidsequences of KshA and KshB or parts thereof having variations of thesequence while still maintaining functional characteristics, areincluded in the invention.

The variations that can occur in a sequence may be demonstrated by (an)amino acid difference(s) in the overall sequence or by deletions,substitutions, insertions, inversions or additions of (an) amino acid(s)in said sequence. Amino acid substitutions that are expected not toessentially alter biological activities, have been described. Amino acidreplacements between related amino acids or replacements which haveoccurred frequently in evolution are, inter alia Ser/Ala, Ser/Gly,Asp/Gly, Asp/Asn, Ile/Val (see Dayhof, M. D., Atlas of protein sequenceand structure, Nat. Biomed. Res. Found., Washington D.C., 1978, vol. 5,suppl. 3). Based on this information Lipman and Pearson developed amethod for rapid and sensitive protein comparison (Science, 1985, 227,1435-1441) and determining the functional similarity between homologouspolypeptides. It will be clear that also polynucleotides coding for suchvariants are part of the invention.

The polypeptides according to the present invention also includepolypeptides comprising an amino acid sequence encoded by thenucleotides 499-1695 of SEQ ID NO:1 or of the nucleotides 387-1427 ofSEQ ID NO:2, respectively, but further polypeptides with a similarity ofat least 70%, preferably 80%, more preferably 90%, and even morepreferred 95%. Also portions of such polypeptides still capable ofconferring biological effects are included.

Another aspect of the present invention are genetically modifiedmicroorganisms. For the construction of mutant strains incapable of9α-hydroxylation, the genes encoding KSH activity must be identified andtheir nucleotide sequence must be known. The two genes of thisinvention, designated kshA and kshB, were identified in Rhodococcuserythropolis SQ1 to encode KSH. These genes were cloned by functionalcomplementation of two UV mutants, designated strains RG1-UV26 andRG1-UV39, both impaired in 9α-hydroxylation of AD(D). They were isolatedvia an extensive screening of UV irradiated cells of R. erythropolisstrain RG1 (van der Geize, R. et al. FEMS Microbiol. Lett. Submitted2001), a kstD (encoding 3-ketosteroid Δ¹-dehydrogenase=KSTD1) genedeletion mutant of strain SQ1. Strains RG1-UV26 and RG1-UV39 were unableto grow on AD and ADD, but grew normally on 9OHAD, indicating deficientKSH activity.

For functional complementation of the KSH deficient mutant strainsRG1-UV26 and RG1-UV39 and cloning of the kshA and kshB genes, a genomiclibrary of R. erythropolis RG1 was constructed using Rhodococcus-E. colishuttle vector pRESQ (FIG. 1). Sau3A digested chromosomal DNA of R.erythropolis RG1 was sized by sucrose gradient to 6-10 kb and ligatedinto BglII digested pRESQ. Transformation of E. coli Top10F′ (InvitrogenCorp.) with this ligation mixture generated a gene library ofapproximately 15,000 transformants in which approximately 90% of theconstructs contained insert. An average insert size of 6 kb wasestimated. No complications with stability or rearrangements wereapparent. The gene library represents the complete genome (p>0.99)assuming a genome size of approximately 6 Mb.

Introducing the R. erythropolis RG1 genomic library into strainsRG1-UV39 and RG1-UV26 and subsequent screening for complementation ofKSH deficiency, resulted in the cloning of two independent DNA fragmentscontaining the kshA gene and the kshB gene, respectively (FIG. 2).

Analysis of these genes revealed that kshA encodes a 398 amino acidprotein (KshA). KshA showed high similarity (58% identity; 84%similarity) to a hypothetical protein encoded by gene Rv3526(DDBJ/EMBL/GenBank accession no. CAB05051) in Mycobacterium tuberculosis(Cole, S. T. et al. Nature (1998) 393: 537-544). Rv3526 is thus expectedto be the homologue of kshA in M. tuberculosis. Comparison of theobtained nucleotide sequence of kshA to databases further revealed thatkshA is identical (97%) to a hypothetical gene (ORF12), found by Maeda,M. et al. (Appl. Environ. Microbiol. (1995) 61:549-555) in R.erythropolis strain TA421 (DDBJ/EMBL/GenBank accession no. D88013)upstream of bphC1 (FIG. 3). In analogy with the molecular organizationfound in strain TA421, a hypothetical ORF11, identified in this straindownstream of ORF12, was also identified downstream of kshA in strainSQ1. The nucleotide sequences of the DNA fragments of strain SQ1 andstrain TA421 were therefore merged and the resulting theoreticalnucleotide sequence was used for the successful construction of plasmidpKSH126 (FIG. 3) used for the introduction of an unmarked in-frame kshAgene deletion in R. erythropolis strain SQ1 (rendering strain RG2) andstrain RG8 (rendering strain RG9).

The kshB gene encodes a 346 amino acid protein (KshB). Databasesimilarity searches revealed that KshB showed high similarity toferredoxin reductase components of multi-component oxygenases. Highestsimilarity (56% identity; 85% similarity) was found with Rv3571 of M.tuberculosis (DDBJ/EMBL/GenBank accession no. A70606).

Inactivation of kshA or kshB by unmarked gene deletion rendersmolecularly defined and genetically stable mutant strains capable ofselective Δ¹-dehydrogenation of AD producing ADD that is not furthermetabolized due to absence of KSH activity (see WO 01/31050). Using thesacB counter selection system (described in WO 01/31050) three unmarkedgene deletion mutant strains were constructed: a kshA mutant R.erythropolis RG2 using pKSH126 (FIG. 3), a kshB mutant R. erythropolisRG4 using pKSH212 (FIG. 4) and a kstD kstD2 kshA mutant R. erythropolisRG9. Strains RG2 and RG4 are derived from R. erythropolis SQ1. StrainRG9 is derived from kstD kstD2 mutant R. erythropolis RG8 using pKSH126(FIG. 3). Strain RG8 lacks both 3-ketosteroid Δ¹-dehydrogenaseisoenzymes (KSTD1 and KSTD2; described in WO 01/31050).

Thus, another aspect of this invention is a microorganism blocked in3-ketosteroid 9α-hydroxylase activity characterized in that it is agenetically modified microorganism, in particular of the family ofActinomycetales, preferably of the Rhodococcus genus and most preferredof Rhodococcus erythropolis. Also preferred is a strain, wherein atleast one gene encoding 3-ketosteroid Δ¹-dehydrogenase activity isinactivated, preferably by unmarked gene deletion. In particularpreferred are the strains RG2, RG4 and RG9.

Also an aspect of the present invention is a method to construct agenetically modified strain of a steroid degrading microorganism lackingthe ability to degrade the steroid nucleus, the method comprisinginactivation of the genes encoding KSH-activity, preferably the genekshA and/or the gene kshB. Preferably, the inactivation of the gene(s)is accomplished by targeted, preferably unmarked, gene deletion.

A further aspect of the present invention is the use of a geneticallymodified microorganism in steroid Δ¹-dehydrogenation, in particular inthe preparation of 1,4-androstadiene-3,17-dione and prednisolone.Preferably, the microorganism for such use has been obtained by targetedgene inactivation, preferably unmarked gene deletion, of the genesencoding KSH-activity in a microorganism of the family ofActinomycetales, preferably the gene kshA and/or the gene kshB.Preferred microorganism for this use is selected from the geneticallymodified strains RG2, RG4 and RG9.

The micro-organism strains Rhodococcus erythropolis RG2, RG4 and RG9have been deposited at the Deutsche Sammlung von Mikroorganismen undZellkulturen GmbH (DSMZ), Mascheroder Weg 1b, D-38124 Braunschweig,Germany under the accession numbers DSM 14544, DSM 14545 and DSM 14546,respectively. These deposits have been made under the terms of theBudapest Treaty.

Methods to construct vehicles to be used in the mutagenesis protocol arewell known (Sambrook et al., Molecular Cloning: a Laboratory Manual,Cold Spring Harbor Laboratory Press, Cold Spring Harbor, latestedition). Furthermore, techniques for site directed mutagenesis,ligation of additional sequences, PCR, sequencing of DNA andconstruction of suitable expression systems are all, by now, well knownin the art. Portions or all of the DNA encoding the desired protein canbe constructed synthetically using standard solid phase techniques,preferably to include restriction sites for ease of ligation.

Modifications and variations of the method for introducing disruptedgene mutations, targeted gene inactivation, and in particular unmarkedgene deletion as well as transformation and conjugation will be obviousto those skilled in the art from the detailed description of theinvention. Such modifications and variations are intended to come withinthe scope of present application.

A person skilled in the art will understand how to use the methods andmaterials described and referred to in this document in order toconstruct micro-organisms lacking KSH activity.

The following examples are illustrative for the invention and should inno way be interpreted as limiting the scope of the invention.

LEGENDS TO FIGURES

FIG. 1. The pZErO-2.1 (curved thin line) based Rhodococcus-E. colishuttle vector pRESQ used for constructing a genomic library of R.erythropolis RG1. rep: 2.5 kb region of pMVS301 coding for autonomousreplication in Rhodococcus sp (thick curved line). lacZ-ccdB: marker forpositive selection in E. coli. aphII: kanamycin resistance marker forselection in Rhodococcus and E. coli.

FIG. 2. Strategy for the separate cloning and identification of the kshAand kshB genes encoding KSH activity in R. erythropolis SQ1 byfunctional complementation of UV mutant strains RG1-UV39 (A) andRG1-UV26 (B), respectively, using several pRESQ derived constructs.

FIG. 3. (A) Overview of the 2.6 kb DNA fragment of R. erythropolis SQ1encoding kshA and its 4.1 kb counterpart in R. erythropolis TA421. Thegrey bar of ORF12, together with the black arrow (proposed size of ORF12by Maeda et al. (1995)), represents the actual size of ORF12 in R.erythropolis TA421, which is identical (97%) to kshA. The “X” indicatesthe point of merger of the two sequences. (B) Scheme of thetheoretically merged nucleotide sequences of the DNA fragments of strainSQ1 and strain TA421, and its use in the construction of plasmid pKSH126for unmarked in-frame kshA gene deletion in strain SQ1 and strain RG8.Numbers 14 indicate the primers used to obtain PCR products 1 and 2 forthe construction of plasmid pKSH126 used for kshA unmarked gene deletionin parent strain SQ1 and kstD kstD2 mutant strain RG8.

FIG. 4. Cloning scheme for the construction of plasmid pKSH212 used inthe construction of kshB unmarked gene deletion mutant strain RG4 fromparent strain SQ1.

EXAMPLES

General

Construction of the pRESQ Shuttle Vector.

A pZErO-2.1 (Invitrogen Corp. San Diego, Calif.) derivative wasconstructed in which the BamHI site was replaced by a BglII site (FIG.1). A SacI-StuI fragment of pZErO-2.1, containing the lacZ-ccdB gene,was duplicated by PCR using a mutagenic forward primer (5′ACCGAGCTCAGATCTACTAGTAACGGC 3′, SEQ ID NO:3), containing the desiredBglII restriction site (double underlined) and a Sac1 restriction site(underlined), and reverse primer (5′ ATTCAGGCCTGACATTTATATTCCCC 3′, SEQID NO:4) with a Stu1 restriction site (underlined). The obtained PCRproduct was digested with restriction enzymes SacI and StuI and ligatedin SacI-StuI digested pZErO-2.1 (pRES14). The aphII gene from pWJ5(Jager, W. A. et al. (1992). J Bacteriol 174:5462-5465) was cloned asblunted HindIII-BamHI fragment (Klenow fill in) into EcoRV digestedpBlueScript (II) KS to construct pBsKm1. The unique BglII site in pBsKm1was destroyed by BglII digestion followed by Klenow fill in (pRES4). Theampicillin cassette present in pRES4 was removed by self ligationfollowing BspHI digestion (pRES9). A 2.5 kb BglII-XbaI fragment ofpMVS301 (Vogt-Singer, M. F. et al., (1988) J. Bacteriol. 170:638-645),containing the region for autonomous replication in Rhodococcus sp., wassubsequently ligated into BamHI-XbaI digested pRES9 to construct pRES11.Construction of pRESQ (6.55 kb) was completed by ligating a 4.15 kbBspHI/NcoI fragment of pRES11 into BspHI-NcoI digested pRES14.

Example 1

Inactivation of Steroid 9α-Hydroxylase Activity by UV Mutagenesis.

Late exponential phase R. erythropolis RG1 (=kstD mutant) cells (2·10⁸CFUs·ml⁻¹) grown in 10 mM glucose mineral medium were sonicated for ashort period of time to obtain single cells. Diluted (10⁴) samples werespread on glucose mineral agar medium and irradiated for 15-20 sec witha UV lamp (Philips TAW 15W) at a distance of 27 cm, on average resultingin 95% killing of cells. After 4 days of incubation, colonies that hadappeared were replica plated on AD (0.5 g·l⁻¹ solubilized in DMSO (50mg·ml⁻¹)) mineral agar medium. A screening for AD(D) growth deficientmutants of R. erythropolis RG1 able to grow on 9OHAD mineral mediumyielded 2 mutants that were clearly impaired in the KSH reaction. Thesemutants, designated strain RG1-UV26 and strain RG1-UV39, showed nogrowth after 3-4 days with either AD or ADD as sole carbon and energysource, while growth on 9OHAD mineral agar medium was normal.

Example 2

Cloning and Molecular Characterization of kshA and kshB.

The R. erythropolis strain RG1 gene library was introduced into strainRG1-UV39 by electrotransformation to complement its mutant phenotype(FIG. 2). A clone was isolated containing a 6 kb insert (pKSH101) thatwas able to restore growth of strain RG1-UV39 on AD mineral agar medium.Restriction enzyme mapping analysis, subcloning in pRESQ and subsequentcomplementation experiments resulted in identification of a 1.8 kbBamHI-Sau3A DNA fragment (pKSH106) that was still able to complementstrain RG1-UV39 (FIG. 2). This 1.8 kb insert was cloned into pBlueScript(II) KS and its nucleotide sequence determined. Nucleotide sequenceanalysis revealed a single 1,197 nt ORF (kshA, 499-1695 of SEQ ID NO:1)encoding a putative protein of 398 aa (KshA).

Complementation of R. erythropolis RG1-UV26 with the strain RG1 genelibrary resulted in isolation of clone pKSH200 able to restore growth ofstrain RG1-UV26 on AD mineral agar medium (FIG. 2). By subsequentrestriction mapping analysis, subcloning and complementation experimentsof pKSH200 we identified a 2.8 kb BglII-Sau3A fragment (pKSH202) whichwas still able to restore the mutant phenotype of strain RG1-UV26 (FIG.2). This fragment was subcloned into pBlueScript (II) KS and itsnucleotide sequence was determined. The ORF responsible forcomplementing the RG1-UV26 mutant phenotype was identified from asubsequent complementation experiment. An Asp718 restriction site mappedon the 2.8 kb fragment was used to construct pKSH205, which could nolonger complement the mutant phenotype (FIG. 2). The Asp718 restrictionenzyme thus is located within the ORF responsible for complementation.The identified ORF of 1,041 nt was designated kshB (387-1427 of SEQ IDNO:2; GC content, 62.3%) encoding a putative protein of 346 amino acidswith a calculated molecular weight of 37.1 kDa (KshB).

Example 3

Unmarked Gene Deletion of kshA in R. erythropolis SQ1.

For unmarked in-frame gene deletion of kshA (ΔkshA) pKSH126 wasconstructed. A 1.3 kb fragment (PCR product 1) was obtained from pKSH101using a primer (FIG. 3 primer 1) annealing to sequences upstream of kshA(5′CGCGGGCCCATCGAGAGCACGTT 3′, SEQ ID NO:5), and a primer (FIG. 3 primer2) annealing to the 5′-end of the kshA gene(5′GCGCCCGGGTCCGAGTGCCATGTCTTC 3′, SEQ ID NO:6) containing a SmaI site(underlined). Primers for PCR product 2 were developed using thenucleotide sequence of the merged sequences of strain SQ1 and strainTA421 (DDBJ/EMBL/GenBank accession no. D88013). PCR product 2 (840 bp)was obtained from SQ1 chromosomal DNA using forward primer (FIG. 3primer 3) annealing to the 3′ end of the kshA gene (5′GCGCCCGGGACAACCTCCTGATTCGCAGTC 3′, SEQ ID NO:7), including a SmaIrestriction site (underlined), and reverse primer (FIG. 3 primer 4)annealing to ORF11 (5′ GCGTCTAGAGTGGAAGAGCATTCCCTCGCA 3′, SEQ ID NO:8),including a XbaI restriction site (underlined). The SmaI restrictionsite was introduced to give an in-frame deletion of kshA followingligation of this PCR fragment behind the 5′-truncated kshA gene from PCRproduct 1. Finally, a 2.05 kb SalI-XbaI fragment from pKSH125 wasligated into pK18mobsacB vector (pKSH126).

Unmarked in-frame deletion of the kshA gene was obtained by introducingthe mutagenic vector pKSH126 in strain SQ1 followed by sacB counterselection (WO 01/31050). Wild type kshA gene was reduced to an ORF(ΔkshA) of 30 nt, encoding only 9 amino acids (MALGPGTTS). Gene deletionof kshA was confirmed by Southern analysis of BamHI digested chromosomalDNA using the 2 kb insert of pKSH126 as a probe: a 2.05 kb wild typeBamHI DNA fragment was reduced to 0.88 kb in the gene deletion mutantstrains. The resulting strain is designated R. erythropolis RG2.

Example 4

Unmarked Gene Deletion of kshB in R. erythropolis SQ1.

For unmarked in-frame gene deletion of kshB construct pKSH212 was made(FIG. 4). For this purpose pKSH201 was fused to a PCR product (1,275 bp)obtained from pKSH202 as a template. PCR product was obtained withforward primer 5′ GCGGGTACCGATCGCCTGAAGATCGAGT 3′ (SEQ ID NO:9) andreverse primer 5′ GCGAAGCTTGCCGGCGTCGCAGCTCTGTG 3′ (SEQ ID NO:10) andligated (blunt) into EcoRV digested pBlueScript(II) KS (pKSH210). At the5′-terminal end of this PCR product an Asp718 restriction site,preceding the stop codon of kshB (see forward primer), was introduced toensure proper in-frame deletion of kshB after ligation with the Asp718restriction site of pKSH201. At the 3′-terminal end a HindIIIrestriction site (see reverse primer) was added, compatible to theHindIII restriction site of pKSH201, for cloning purposes. PCR productwas isolated from pKSH210 by Asp718-HindIII digestion (1,263 bp) andligated into Asp718-HindIII digested pKSH201 (FIG. 2) (pKSH211), therebyintroducing the desired kshB in-frame deletion (FIG. 4). Finally, aBamHI-HindIII DNA fragment (2.2 kb) containing the kshB deletion wasligated into pK18mobsacB (see WO 01/31050) digested with BamHI andHindIII (pKSH212).

Plasmid pKSH212 was introduced into R. erythropolis SQ1 by conjugationusing Escherichia coli S17-1. Unmarked kshB gene deletion was obtainedusing the sacB counter selectable system (WO 01/31050). Potential kshBmutants were screened by replica plating on AD mineral agar plates,which enabled us to isolate kshB mutants unable to grow on AD. Southernanalysis was performed on Asp718 digested chromosomal DNA of wild typeand three AD growth deficient mutants. Hybridization with the completekshB gene showed that kshB was not present in the genome of the putativekshB mutants. A clear hybridization signal (4.3 kb fragment) wasexclusively found with wild type chromosomal DNA. Additional Southernanalysis with an alternative probe, being the 2.2 kb insert of pKSH212comprising both flanking regions of kshB, furthermore confirmed kshBgene deletion: a 4.3 kb Asp718 wild type DNA fragment containing thekshB gene was reduced to 3.3 kb in a kshB mutant, demonstratingreplacement of the 1,041 bp kshB gene by a kshB in-frame remnant of 30nt (encoding MTTVEVPIA). The resulting strain is designated R.erythropolis RG4.

Example 5

Use of a Genetically Modified Strains RG1-UV26, RG1-UV39, RG2, RG4 andRG9 in steroid Δ¹-dehydrogenation.

Strains RG2 and RG4 were plated on mineral agar media containing AD, ADDor 9OHAD as sole carbon and energy source. Both strains showed no growthon AD(D), whereas growth on 9OHAD was comparable to strain SQ1. Thesephenotypes are in agreement with those found with UV mutant strainsRG1-UV26 and RG1-UV39. Bioconversion of AD (1 g·l⁻¹) with strain SQ1results in AD utilization but not in accumulation of ADD or othermetabolites. Bioconversion of AD (1 g·l⁻¹) by strain RG2 or strain RG4resulted in comparable accumulation levels of ADD (varying between0.3-0.5 g·l⁻¹ after 168 h). AD(D) 9α-hydroxylation thus is blocked byinactivation of either kshA or kshB, demonstrating the essential role ofboth KshA and KshB in KSH activity in R. erythropolis SQ1. In ADbioconversion experiments with strain RG9, neither a decline in theinitial AD concentration nor 9OHAD formation was observed. Mutant strainRG9 thus confirms that kshA encodes AD 9α-hydroxylase activity and that,contrary to the KSTD isoenzymes, no further KSH isoenzymes are presentin R. erythropolis SQ1.

1-16. (canceled)
 17. A method of steroid Δ¹-dehydrogenation, comprisingproviding the steroid to a genetically modified microorganism blocked in3-ketosteroid 9α-hydroxylase activity by inactivation of at least onegene encoding KSH-activity.
 18. The method of claim 17, wherein steroidΔ¹-dehydrogenation is used in the preparation of1,4-androstadiene-3,17-dione and prednisolone.
 19. The method of claim17, wherein the microorganism is of the family of Actinomycetales. 20.The method of claim 19, wherein the microorganism is of the Rhodococcusgenus.
 21. The method of claim 17, wherein the microorganism further hasat least one gene encoding 3-ketosteroid Δ¹-dehydrogenase activityinactivated.
 22. The method of claim 17, wherein the inactivation of thegene(s) is accomplished by targeted gene deletion.
 23. The method ofclaim 22, wherein the inactivation is accomplished by unmarked genedeletion.
 24. The method of claim 21, wherein the inactivation of thegene(s) is accomplished by targeted gene deletion.
 25. The method ofclaim 24, wherein the inactivation is accomplished by unmarked genedeletion.
 26. The method of claim 17, wherein the microorganism isselected from the genetically modified microorganisms RG2, RG4 and RG9.27. The method of claim 17, wherein the gene encoding KSH-activity isselected from the group consisting of the gene kshA and the gene kshB.